STEM CELL THERAPY: THE POTENTIAL IMPORTANCE OF RESEARCH INTO "THERAPEUTIC CLONING", EMBRYONIC STEM CELLS AND ADULT STEM CELLS.

Robin Lovell-Badge. Division of Developmental Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

We have now become very used to the idea of organ transplants in medicine, for a wide range of problems from cataracts to kidney or heart failure. However, we are also all aware of the frequency with which they fail. Immune rejection is one of the most common causes of graft or transplant failure (contaminating pathogens being another major problem). There is also a serious shortage of donors.

What can we do about this ? Both problems could be solved if autologous grafts are performed, taking tissue from one part of the body to repair another. But there are relatively few cases where this can be done at present - skin grafts for burns victims, or valves from leg veins used to repair heart valves.

Rather than using whole organs or tissues, an alternative would be to isolate and use special cells called stem cells. In fact we already do this with skin grafts as mentioned above, or with bone marrow transplants, where the stem cells in the bone marrow can regenerate all the different types of cell in the blood. However, we are all aware how difficult it is to find the correct tissue match to do this. The ideal source being an identical twin, which few of us have. But there are many other types of stem cell. Could these be used for therapy ?

First, how do we define a stem cell ? When a mature or differentiated cell divides, it can only give rise to the same type of cell. However, when a stem cell divides, it gives rise to another stem cell (i.e. self renew) and to a cell that is differentiated. The latter may still be able to divide, but it can not go back to form the original type of cell (see Fig. 1).

There are many different types of stem cell and these are present at all stages from the early embryo to the adult. Stem cells that are present in the embryo, tend to divide frequently, and many have the potential to give rise to a wide range of more specialised cell types. They are therefore considered as multipotent stem cells (Fig. 1).

Adult stem cells are present in many tissues, but they are often quite rare and divide infrequently. They also tend to have limited potential, indeed, stem cells have not been recognised for many cell types in the adult. For example, those that would give rise to the cells of the lung epithelium that are defective in Cystic Fibrosis. Where adult stem cells do occur, they are usually in tune with the organ to which they belong, dividing at the appropriate rate to both self renew and give rise to just sufficient differentiated cell types to replenish those that have been lost. For example, in the blood, skin or brain. However, with accidental trauma or disease the normal rate of regeneration is often too low to allow repair. This is particularly true within the nervous system, but also in other tissues where the normal rate of turnover is low, such as the pancreas.

The idea behind stem cell therapy, is to isolate such cells, multiply them in vitro and then use them to replace damaged tissue. This is exactly as is done to repair skin in burns victims. However, many more types of disease could be treated in this way than with conventional organ transplants, as often it is one cell type that has gone wrong rather than the whole organ. It may also be particularly suitable for chronic debilitating diseases, such as Parkinson's, Multiple Sclerosis, diabetes, etc.

However, in many of these diseases, as well as in cases where there is more acute organ failure, it may be too difficult to isolate the appropriate stem cells from the affected tissue in the patient. The stem cells may already be defective, too rare or no longer present at all. Even if they could be isolated, they may not be able to grow well enough in vitro to give sufficient numbers of cells for therapy.

Each type of adult stem cell was thought to be able to give rise only to its normal range of mature cell types. However, recent, very exciting work has now suggested that, in some circumstances, it is possible for one type of stem cell to change into another. For example, for a blood stem cell to give nerves. This might allow patient-specific stem cell therapy, where stem cells from one part of the body are used to repair damage to another. I will return to this later.

The stem cells with the greatest potential, however, are the so-called, Embryonic Stem cells. These are derived from an early preimplantation embryo stage called a blastocyst. Figure 2 shows a mouse blastocyst, which corresponds to about 3 days of development post fertilisation. This is similar to a human blastocyst at 5 days of development. At this stage there are a maximum of 100 cells, comprising just two cell types. The outer ones will form part of the placenta (trophoblast), the inner cells (inner cell mass or ICM) will form the embryo itself, although much later, after implantation. In fact, even though cells of the ICM may each have the potential to contribute to the embryo proper, the majority of them will give rise to other components of the placenta and to membranes such as the amnion, yolk sac, allantois and umbilicus. These all provide the support system for the embryo, but are discarded at birth. In fact, it is not possible to point to a single cell at the blastocyst stage and say that this will contribute to the newborn animal or person. The cells do not know what they will become, therefore we can not possibly know.

Both cell types in the blastocyst depend on each other for their proper development and survival. Thus the inner cells will not develop into an embryo if they are removed from the outer cells. However, they can be grown in a petri dish where, at high frequency, they give rise to Embryonic Stem cells (Fig. 2, centre).

Embryonic Stem cells have a number of remarkable properties. They can essentially be grown indefinitely, and in very large numbers. However, they are not "transformed" like other permanent cell lines. (Transformed means that they have undergone some mutation in a gene that allows permanent growth or they carry a transforming gene from a virus or tumour that has the same effect.) Embryonic Stem cells are normal cells by all criteria, such as their chromosome make up (karyotype). They also have the ability, under the right conditions, to give rise to all cell types of the body. The best test of this in the mouse is to inject them into a blastocyst, where they reintegrate into the ICM and can contribute to all tissues in the resulting chimaera. Such chimaeric mice can live a normal life, with no greater incidence of tumours, etc than found in the strains of mice used to make them.

However, Embryonic Stem cells can also form a wide range of cell types in vitro. We already know how to direct them to form certain types of cell purely in culture (see Fig. 2, right panel). For example, nerve cells, muscle cells, cells that form blood vessels, pancreatic islet cells. Cells made like this can then be grafted back into animals, where they have been shown to at least partially correct a range of diseases. These include mouse models of Parkinson's Disease, myelin-deficient (md) rats (a good animal model for the hereditary human myelin disorder Pelizaeus-Merzbacher disease, which has some relevance to multiple sclerosis) and diabetes. (See further discussion below and Table 3.)

We have been studying Embryonic Stem (ES) cells in the mouse for a long time, at least 20 years, so we know a lot about them. But since the work of Jamie Thompson and his colleagues two years ago, we now know it is possible to make them from human blastocyst stage embryos as well. They clearly share many of the properties possessed by mouse ES cells, including the ability to make many different cell types in culture.

So, could we use human ES cell lines to treat any of a wide range of diseases, by cell-based therapies ? It would seem likely, except for the big problem of immune rejection. The few lines that have been established so far (in other countries), would not overcome this problem. One possibility is to have available many lines, perhaps a bank of 100 or even 1000 or so, which would be tissue-typed, so that a close enough match would be available for most patients and for most types of therapy. This would still require the use of immunosuppressive drugs to prevent rejection of grafted cells, which can have a number of more or less severe consequences to the patient, including increased likelihood of infections or (possibly) tumours. These consequences would have to be weighed against the likely benefit of the therapy. Some types of graft are known to require closer tissue matches than others - bone marrow grafts being the most extreme case (partly because there is the possibility that the graft can reject the host, as well as the usual problem of the host rejecting the graft). However, other potential cell-based therapies may also require very close tissue-matching. For example, many cases of diabetes occur when the b-cells in the islets of the pancreas, which are the cells that make insulin, have been destroyed by the patients own immune system.

The ideal option would be to isolate Embryonic Stem cells from the patient, but of course the right cell type to do this only exists within the very early embryo. What if we could reverse the normal direction of differentiation and obtain suitable stem cells from an adult cell ? The nuclear transfer (cloning) techniques, that gave rise to Dolly and subsequently to cloned mice, cows, goats and pigs, showed that it is indeed possible to reprogramme the nucleus of an adult cell.

The rationale behind these experiments was that the most likely source of a factor that could reprogramme an adult cell to become an embryo cell, would be from an early embryo itself or from the cells that normally have the potential to give rise to an embryo. These are the germ cells, which are present as unfertilised eggs in the female and as sperm in the male. It is a great pity, but we can not use sperm to reprogramme. They are present in vast numbers, but they are useless. Some would argue that they are typically male ! As usual, we have to turn to women to provide the solution.

The unfertilised egg (or oocyte), which, apart from carrying one set of chromosomes containing the genetic information in DNA, has a large amount of cytoplasm. This contains the factors that normally reprogramme the incoming sperm nucleus into one appropriate for an early embryo (Fig. 3). It also turns out that this cytoplasm can reprogramme an adult cell nucleus, essentially tricking it into "thinking" that that it is the nucleus of a one-cell embryo. So, by removing the oocyte's own nucleus and replacing it with that of an adult donor cell, it is possible to obtain an embryo. This is the nuclear transfer technology that allows cloning. Many experiments have shown that an early embryo cell has already lost the ability to reprogramme a transferred nucleus, suggesting that the relevant factors are no longer present once embryonic development is initiated.

It should be absolutely clear, however, that we are not talking about reproductive cloning. This would require that the preimplantation embryo was allowed to develop in the womb (uterus) of a surrogate mother, something that is illegal. It is very easy to draw a strong bright line between development in culture to the blastocyst stage and subsequent development within the uterus. The latter requires so many additional hurdles to be overcome that it is likely to very impractical and it would certainly be very hard to break the law. (In the UK it would require the consent of the egg donor, the clinicians and embryologists willing to perform the procedures, the consent of the surrogate mother (or probably many) and a licence from the HFEA. The latter have made their position on this very clear: they would not approve it.) Instead, we need to focus on the topic we are debating, namely, can we use this nuclear transfer technology to derive human Embryonic Stem cells that can be used for therapeutic purposes ? And, could it work well enough to be done on a patient-specific basis, to overcome problems of immune-rejection ? The simple answer is we do not know and this is one of the reasons why research in this area is necessary. While the nuclear transfer techniques have worked in a handful of very different species, there are others for which it has so far been unsuccessful (e.g. rats, rabbits and monkeys). We do not know the situation for humans.

We should consider again the two potential ways of obtaining suitable stem cells for cell-based therapies. These are illustrated in Fig. 4 for the ideal case of patient-specific stem cell therapy, but mostly apply also to the establishment of banks of stem cells from a suitably large and diverse range of individuals or early embryos left over from IVF programmes.

The idea of therapeutic cloning or cell-nuclear replacement technology is shown on the right hand side of Fig. 4. A biopsy would be taken from the patient and by nuclear transfer, reprogramme an adult cell into an early embryo. This would be cultured in vitro to the blastocyst stage and then the inner cells isolated and used to derive Embryonic Stem cells. These would have the same genetic make-up as the patient. Indeed, they can be considered an extension of the patient. We could then apply techniques, many of which we have learned from studying mouse Embryonic Stem cells, to direct these to form the relevant cell type to cure the patient, be it Parkinson's, heart disease or spinal cord injury. Indeed, recent work has already suggested that it is possible to direct human ES cells to differentiate along specific pathways. If there is a genetic cause to the disease, such as cystic fibrosis or muscular dystrophy, it may be possible to correct the genetic defect in the stem cells, prior to grafting the cells back into the patient. Techniques for doing this are well established with mouse ES cells. Clearly, once Embryonic Stem cells have been made for an individual, they would be available for treating any other problem present in that person.

We do not know the best source of cells for the original biopsy. Several cells types have worked in other species, including the tip of the tail in mice, which is essentially a skin biopsy. But this is another area where research is needed. It is possible that some easily accessible human cell types make particularly good nuclear donor cells.

The second way we might be able to obtain a wider range of stem cells for therapy is by redirecting one type of adult stem cell into another. This is illustrated on the left of Fig. 4. In this case, one type of stem cell would be isolated and then reprogrammed into another type of stem cell, appropriate for curing the disease. For example, blood stem cells could be turned into nerve stem cells and then into the appropriate nerves for e.g. curing Parkinson's.

So, one might ask, if we can do this, then why would we need to even think of using the first method ? I think this is best illustrated by looking at some of the factors that we need to consider before we can attempt to do stem cell therapy, and to compare and contrast the two methods.

Table 1 lists some of the requirements for stem cell therapy.

1. Accessibility.

Adult stem cells are often very rare and inaccessible. Blood stem cells are present at very low numbers in blood (about 1 in 10,000,000). They are more frequent in bone marrow, but still rare, and bone marrow biopsy is an operation with some risk and discomfort. Cord blood, obtained from the umbilicus at birth is another source of blood (hematopoietic) stem cells. These need to be banked and stored in anticipation of any disease. Although this is already being done in some centres, it obviously can not be done retrospectively. Furthermore, in over 1500 publications involving the study or use of cord blood stem cells, so far no paper describes their purification, and there is no indication that they can give rise to any cells other than those typical of the haematopoeitic system. The stem cells of the pancreas, which can give islet cells, are thought to be in the pancreatic ducts, but methods of isolating them so that they can be expanded in vitro have not been found. Grafts of islets have been shown to work, but for each patient there is a requirement for multiple donors and aggressive immunosuppression. With respect to neurons and glia, stem cells do exist in the brain and spinal cord, but these central nervous system or CNS stem cells are rare and tend to be in regions that are difficult to access safely (see Fig. 5). Clearly one would not want to damage CNS tissue getting them out. There is also concern about grafting CNS material from one individual to another, not only because of the possibility of rejection, but also because of hidden diseases such as CJD.

For many adult cell types we do not know where the stem cells are or if they even exist. For example, no one has identified stem cells that would replace the cells that line the lung, needed in cases of Cystic Fibrosis, emphysema or after inhalation burns. On the other hand, we know that Embryonic Stem cells are able to give rise to all cells of the lung in chimaeric mice, so it is likely that they would do so in vitro. With an appropriate marker it may be possible to select them and indeed to discover the cellular pathway that leads to their formation. Such research may allow adult lung stem cells to be found.

To make human Embryonic Stem cells, we need to consider either the use of spare embryos from IVF programmes or the use of nuclear transfer to reprogramme an adult cell. Clearly the number of spare embryos is limiting. However, many more are currently discarded than would be needed to establish say 1000 ES cell lines over a period of a few years. Even from the few studies done so far, it seems that the frequency of deriving human ES cells from normal blastocysts is very high (25-33%). Indeed it may even be better than with mice, where it very strain dependent - as high as 80% with one particular strain (129SV/EV), but down to almost zero with most others. (N.B. There have been no apparent genetic background effects so far in deriving human ES cells: the frequencies were similar with blastocysts obtained in North America and in Singapore. With more experience it may be possible to improve the efficiency of this step even more, but it already seems to be one that is not worth worrying about too much.

For some people, who believe that life begins at fertilisation, the use of spare embryos left over from IVF programmes to derive Embryonic Stem cell lines would be unacceptable. However, the cell nuclear replacement technique ("therapeutic cloning"), uses unfertilised eggs. These do not have the same moral value and, especially as their genetic material is removed and replaced with an adult cell nucleus, they could be considered just as an extension of the adult: a universal organ available specifically for donation ! If made on a patient-specific basis, this would even overcome some religious objections to transplants between individuals, as the graft would essentially be autologous.

With respect to the use of nuclear transfer to reprogramme an adult cell we need to consider both the source of unfertilised eggs and suitable donor cell type. With respect to the former, some people have raised the problem that there would be a shortage of unfertilised eggs to contemplate anything on a large enough scale to have patient-specific stem cell therapy. This is essentially true if it is necessary to use only those leftover from IVF programmes. However, it is possible that only a small fraction of egg cytoplasm is required to reprogramme some adult cells. Ultimately, it may be possible to define and isolate the factors from the egg cytoplasm that are responsible. This is one intention of the research that needs to be done. If successful, this would obviate the need to use any eggs.

It is worth mentioning sources of unfertilised eggs other than those available from IVF programmes. Techniques are being developed in the mouse and in farm animals, such as cattle, to remove from the ovary so called primordial follicles, which contain an oocyte that has not yet begun to grow. These are found at all stages from the foetus to the adult. These follicles can then be maintained in culture in conditions that promote growth of the oocyte. In one study in the mouse, such a follicle was isolated from a newborn female mouse, the oocyte grown and matured in vitro and then fertilised, also in vitro, to obtain an early embryo, which after embryo transfer, gave rise to a normal liveborn mouse. Ethical issues notwithstanding, this raises the possibility of using oocytes derived from the ovaries of aborted fetuses. (This is currently not permitted in the UK.) We also have to consider that in the future women may become more willing to donate unfertilised eggs. To be blunt, some 100,000,000 unfertilised eggs are flushed down the toilet each year in the UK. Of course it would be difficult to retrieve these (again it is a pity that we can not use sperm). But, one should consider how many women would be willing to undergo the superovulation procedures and surgery necessary to provide a sufficient number of unfertilised eggs, if they know that a loved one could benefit from stem cell therapy ? Clearly it would be unethical to put any pressure on a woman to do so - and as a man I have to be careful even putting this idea forward, but it is something that needs to be considered.

With respect to donor cell types, if a skin biopsy can be used, then this would pose no problems in terms of either source or rarity. If other cell types are found to be even better, and one possibility is that adult stem cells may be easier to reprogramme than differentiated cells, then there is still the advantage that very few cells are actually needed for the nuclear transfer step. We know that skin stem cells are one of the few adult stem cell types able to divide extensively, so perhaps these would be ideal nucleus donors. However, they have a limited potential. Blood stem cells may be better, if there are simple, robust methods for their isolation.

2. Properties in vitro

It is often difficult to isolate pure populations of stem cells from adult tissues, they tend to grow poorly and they often have a limited lifespan. All cells age as we grow older and stem cells are no exception. This will include a shortening of telomeres, the special regions at the end of chromosomes required to maintain their integrity. Cellular ageing has been associated with telomere shortening. It will also include the accumulation of mutations (see below). Conditions for growing adult stem cells in vitro have not been established for most stem cell types: CNS and skin being perhaps the two most notable exceptions. Because the stem cells grow slowly and for a limited number of divisions it may be difficult to obtain sufficient numbers for therapy. One possible way to circumvent this problem is to "transform" them with an "immortalising" viral or cellular oncogene (tumour causing gene). This has been used for some studies in the mouse using adult CNS stem cells (see papers by Evan Snyder) and liver (Kobayashi et al, 2000). However, for use in humans there would have to be robust methods for eliminating the oncogene prior to grafting.

Cells that grow slowly tend to be much harder to manipulate or from which to select desired properties. Indeed, somatic mutation occurring in vitro may result in faster growing cells, which will take over the culture. These may have changed in other properties, such that they will no longer be suitable for therapy and indeed they may no longer be safe to use (see below).

With respect to the range of cell types that a given adult stem cell can give rise to, some examples are presented in Table 2. Clearly they can all give rise to the specialised cells that reflect their normal function in vivo, i.e. if they had been left in the organ to which they belong. CNS stem cells can therefore give rise to both nerves (neurons) and to several types of support cell, collectively called glial cells. Within the CNS there are several types of glia, including oligodendrocytes and astrocytes. Schwann cells are the glia of the peripheral nervous system. These may have specific characteristics depending on the neurons with which they interact and the particular region of the nervous system where they are located. With respect to neurons, however, there is a large, very diverse range of cell types. In many of the experiments that have been done so far using adult CNS stem cells, the precise identity of the neurons has not been ascertained. Indeed, some evidence suggests that stem cells isolated from one part of the CNS may only be able to give rise to neurons typical of that region of the CNS. If we have to be even more precise about the source from which they are obtained, this raises further difficulties that will have to be overcome before we can contemplate using adult stem cells for therapy.

Data that has accumulated over the last few years has shown that adult stem cells can give rise to a much greater range of differentiated cell types than expected, even those typical of other tissues. For example, CNS stem cells have been shown to give muscle and blood cell types, while blood (haematopoietic) stem cells can give muscle and liver cells (hepatocytes) and to cells of the CNS (Alison, et al. 2000; . This latter result was described in two recent papers. Adult mouse bone marrow (blood) stem cells when injected into mice that have been irradiated to kill their own blood stem cells or carried a mutation that did so, were able to change their normal fate and give rise to neurons in the injected mice (Brazelton, et al, 2000; Mezey, et al., 2000).

All these results are very exciting from a fundamental scientific point of view and would seem to provide a very useful alternative way to carry out patient-specific stem cell therapy (as outlined in Fig. 4). However, in reality we know almost nothing about the process that allows one adult stem cell type to change into another. We do not know what is responsible for the reprogramming, nor, with a few exceptions, do we how to direct the stem cells to change into any other particular cell type in vitro. Moreover, the change of potential occurs very infrequently and is likely to occur with only a few relatively rare cells amongst the original stem cell population. Are these cells normal and safe ?

Apart from a few special cases, the only situations where this change in adult stem cell fate has been seen so far are when the isolated stem cells have been put back into an animal (or human). There is, therefore, essentially no control over the process. For example, with the recent papers mentioned above, the new neuronal material was of several types, including many fairly unspecialised cells, and there was no evidence that the cells were normal or functional. There can not be any control over the type of neuron produced as they have differentiated in vivo. Moreover, the bone marrow stem cells may only have colonised the CNS because stem cells in this tissue were also destroyed by the irradiation. This would explain contributions to the olfactory system where cell renewal is a normal continuous process. (See the commentary that accompanied these two papers by Vogel, (2000)).

In fact the best example which shows that stem cells from the adult mouse brain can give rise to many other mature cell types, came from studies by a Swedish research group (Clarke et al, 2000), where they could reprogramme the cells by injecting them into blastocyst stage mouse embryos. The resulting embryo chimaeras showed contributions from the stem cells in several tissues including muscle, bone, gut, etc, (although apparently not all cell types could be formed). From a fundamental biological research perspective this was a fascinating result. But, clearly we can not and would not want to use this method of reprogramming human stem cells. It was also a rare event, working in only 1% of attempts. This is in contrast to carrying out the same procedure with ES cells, where essentially 100 % of cells (even single cells) injected into a blastocyst can contribute to many tissues in the resulting chimaera. The authors also described a second method, namely to co-culture the CNS stem cells with differentiating ES cells, but there is so far no understanding of how this worked and relatively little control over the process. Unless the factors responsible can be found, one might as well just use the ES cells.

In contrast, Embryonic Stem cells can be purified very easily, they grow very well in culture and they are essentially immortal without the need for "transformation". There is good evidence that the reprogramming by the cell-nuclear replacement ("cloning") technique also rejuvenates the adult cell nucleus. Although the first studies on Dolly indicated that her telomeres are the same length expected for a 6 year old sheep, studies on cloned mice and cattle are different and reveal that the telomeres are restored to the normal expected length.

We know that Embryonic Stem cells can give rise to any cell type within the body, so potentially they can be used to treat any disease. This also applies to the problem of regional specification as discussed above for adult stem cells: ES cells can give rise to any of the many types of neuron found in any part of the nervous system. In addition, we already know how to select particular cell types from differentiating mouse ES cells and to do this in a controlled manner. There are now many examples, so I list only some, with obvious therapeutic benefits, in Table 3. Most of these have been tested to some extent in animal models of the corresponding human disease or injury. Two recent studies are particularly noteworthy. Firstly, the derivation of a stem cell type able to give rise to all the cell types that make up blood vessels (Yamashita et al, 2000). These would have obvious potential to treat a range of chronic problems, including coronary heart disease. Secondly, through the discovery of a new factor a Japanese team led by Yoshiki Sasai have been able to obtain relatively pure populations of dopaminergic neurons, the cell type that is defective or missing in Parkinson's disease, from ES cells in essentially a one step procedure (Kawasaki et al, 2000). In previous work others have been able to obtain such neurons from ES cells, but only by following a complex method and they comprised only a small fraction of the resulting neuronal cell types. Cells produced by this new method were grafted into the brains of mice that had been chemically depleted of their own dopaminergic neurons, and the grafted cells were shown to produce dopamine. This is clearly a very promising result with respect to potential treatments of Parkinson's disease by cell-based therapies.

3. Requirements for transplant

It is obviously very important to know that any cells used for transplant are safe, free from contamination (pathogens and other cell types) and that they have the desired properties. For Embryonic Stem cells, it is normal for them to give rise to many cell types. They follow a similar progression as found in an embryo. However, for an adult stem cell to change its fate it has to undergo an abnormal process. Indeed, in all cases described so far this has been a very inefficient process. Because the ability to change potential is rare, the cells that are able to do so may in fact be abnormal cells. Tumour cells often have the properties of immature cell types and it is likely that many cancers arise from stem cells that have undergone some mutation. As mentioned above, cells tend to accumulate mutations as they age. Clearly we would not want to risk using cells that might lead to cancer.

If using ES cells it is important to be able to remove any undifferentiated ES cells from the cells required for therapy, otherwise they can form a type of tumour called a teratocarcinoma. However, once they have even begun to develop into a more mature cell type they are known to be very safe. It is also important to transplant back only the desired cell types as others may create scar tissue. Several ways of doing this have been established with mouse ES cells, where the wrong cell type can be eliminated and/or the right cell types selected or purified from a mixed population of cells (e.g. Li et al, 1999; Kawasaki et al, 2000). Again, this is an area where research on human ES cells needs to be carried out.

A large number of studies have been conducted (mostly in mice) with grafts of cells derived from differentiating ES cells and some from adult stem cells. It is difficult to review all of these here. However, the results from studies beginning with ES cells have been very promising, frequently demonstrating good survival of the grafted cells and long term maintenance of function. Moreover many cases have shown at least partial rescue of the disease model.

With adult stem cells, some studies show promising results when they are used to replenish the cell type to which they would normally give rise. However, there is little evidence from any of the studies that have been done so far that involve a change of one stem cell type to another, to suggest that this may be either a useful or a safe therapeutic approach.

Perhaps this just means that we need to do much more research, particularly with adult stem cells. Indeed it is likely that results obtained with ES cells can be applied to adult stem cells and vice-versa. But from all the criteria above, it seems that therapeutic approaches arising from ES cells are likely to lead to successful cures for a far greater range of affected tissue types and far sooner than will be possible with adult stem cells.

Many of these points have been covered already, but it is worth repeating some of them. Clearly much work can and will be done using animals such as the mouse. Indeed, two recent papers, one published (Munsie et al, 2000) and one in Press (Roger Pederson, personal communication), have shown that it is feasible to begin with a biopsy, carry out nuclear replacement into an unfertilised egg, grow embryos to blastocyst stages in culture and then use these to derive Embryonic Stem cell lines. At least some of the latter were shown to have properties expected of ES cells in terms of their ability to differentiate into a wide range of cell types in vitro and in chimaeras.

However, there are many species-specific differences in early development, for example, the rate of development. Furthermore, it is likely that we will need to use human material to reprogramme human cells, especially if they need to follow the normal steps of embryonic development up to the blastocyst stage. This is one reason why it might not be possible to use unfertilised eggs from non-human species. (There are also potential problems about incompatibility between genes in the nucleus and those in the mitochondria in the cytoplasm of the egg.)

We know it is possible at high efficiency to derive Embryonic Stem cells from human blastocysts cultured in vitro from spare embryos obtained in IVF programmes. However, we do not yet know if it is possible to use the nuclear transfer technology to reprogramme adult cells taken from a patient and to obtain suitable blastocyst stage embryos. Nor do we know the best type of cell to use as a nucleus donor. We also need to do research on the best ways to treat any ES cell lines such that we could obtain the right cell type to use for therapy,

It may be that most adult stem cells will be inappropriate and that we need to begin with Embryonic Stem cells to obtain anything useful for cell-based therapies. Promising results from mouse studies have already indicated that cells derived from ES cells in culture can be used to treat a variety of syndromes, such as mouse models of Parkinsons' Disease and diabetes. Do we really want to ignore what seems a very promising method of treating many debilitating diseases ?

We therefore need to begin to explore the usefulness of human ES cells, and as part of this, we need to have methods of quality control to know that any cells used will be safe and reliable. All the evidence obtained with mouse ES cells suggests that they probably will be, but it is important to show this for human ES cells.

We know that Embryonic Stem cells are able to give rise to any cell type within the body. This is a normal process, not one that involves a rare cell changing its potential. They would then seem to be an ideal source of cells for cell-based therapies. However, if they were to be made on a patient-specific basis using current nuclear transfer technology, then this would require the use of many unfertilised eggs, much more than could be reasonably expected to be left over as spare eggs from IVF. However, the reprogramming mechanism elicited by egg cytoplasm after nuclear transfer, is still the only way we know how to reprogram an adult cell in a controlled manner. We need to be able to define the factors that are responsible and work out how to use them or the technology in a more efficient manner. Once we understand the mechanisms, perhaps we could treat adult cells in an appropriate way to directly turn them into the equivalent of Embryonic Stem cells, eventually without having to use any human eggs.

We are dealing with a question of potential risk versus likely benefits. My personal view is that the benefits are definitely worth pursuing. Moreover, it is better that this type of work is permitted in the context of a well regulated and controlled system, such as that already in place in the UK, where the science can proceed in step with ethical issues, rather than in many countries around the world that have little or no regulation.

The following are a selection of references used in preparing this document. These are essentially the same as those cited in the Royal Society Briefing Document, but some extra papers are included. These are highlighted with an asterisk..

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Figure 1.

On the left, when it divides, a typical adult stem cell is shown to give rise to a more mature, or differentiated, cell type as well as a cell similar to the original cell type (self-renewal). On the right, a multipotent stem cell, which is more typical of the embryo, is able to give rise to many types of differentiated cells in addition to self-renewal. The dashed arrow between the two types of stem cell reflects recent evidence that one stem cell type can give rise to another in some circumstances.

Figure 2.

For this figure see the original article in Europäische Akademie where you will find other articles dealing with the science, ethics and aspects of the law in different European countries, around the same topics as in this document.

Left panel: A mouse blastocyst at 3.5 days of development. Cells of the inner cell mass are revealed with a blue stain. Centre panel: Mouse Embryonic Stem cells. Right panel: A pure population of neurons derived entirely in vitro from differentiating Embryonic Stem cells. The inset shows such a neuron after transplantation into a newborn mouse brain (data from Austin Smith).

Figure 3.

The sequence at top shows normal fertilisation of an egg and its development to a blastocyst stage early embryo. The hypothetical factor(s) responsible for reprogramming the incoming sperm nucleus are indicated as red dots. The sequence below shows the nuclear transfer procedure to remove the genetic material from the unfertilised egg and to reprogramme an adult cell nucleus, and subsequent development in culture to a blastocyst, from which ES cells could be derived.

Figure 4.

Patient-specific stem cell therapy. See text for details.

Figure 5.

For this figure see the original article article in Europäische Akademie where you will find other articles dealing with the science, ethics and aspects of the law in different European countries, around the same topics as in this doicument.

The panel on the left shows a mouse embryo at about 14 days of development. The blue stain (which detects the activity of a gene called Sox2) reveals much of the central nervous system. Many of the stained cells correspond to embryonic CNS stem cells. The panels to the right, show adult spinal cord (top) and brain. The blue stain (also detecting Sox2 activity), reveals the regions in which adult CNS stem cells are to be found.

TABLE 1

REQUIREMENTS FOR STEM CELL THERAPY
 
 

TABLE 2

POTENTIAL OF ADULT STEM CELLS
 
 

TABLE 3

SPECIFIC CELL TYPES ISOLATED FROM MOUSE ES CELLS